Abstract
Saccharomyces Rrm3p, a member of Pif1 5'-3' DNA helicase subfamily, helps replication forks traverse protein-DNA complexes, including the telomere. Here we have identified an Rrm3p interaction protein known to be Def1p. In def1 mutants, telomeres were approximately 200-bp shorter than that in wild-type cells. DEF1 is also required for the stable maintenance of mitochondrial DNA, and the telomere shortening phenotype seen in def1 cells is not a secondary consequence of the mitochondrion defect. A combination of DEF1 null mutation with deletion of EST2 or EST3 resulted in an accelerated senescence phenotype, suggesting that Def1p is not involved in the telomerase recruitment pathway. In the absence of telomerase, cells escape senescence by either amplifying Y' regions or TG-telomeric repeats to generate type I or type II survivors, respectively. Only type I survivors were recovered from both def1Delta est2Delta and def1Delta est3Delta double mutant cells, further suggesting that the function of Def1p in telomere maintenance is specific. Our novel findings of the functions of Def1p in telomere and mitochondria suggested that Def1p plays multiple roles in yeast.
Highlights
Telomeres are the physical ends of eukaryotic chromosomes that are composed of specific repeat DNA sequences and binding proteins [1]
Identification of Def1p—To study how telomere replication by conventional replication machinery and telomere elongation by telomerase are connected, an affinity purification of the proteins that interact with Rrm3p was carried out because Rrm3p promotes the replication fork progression through both telomeric and subtelomeric DNA [34]
The affinity chromatography using Rrm3p-13myc as bait and one-dimensional liquid chromatography-MS/MS sequencing allowed us to identify the proteins associated with Rrm3p
Summary
Telomeres are the physical ends of eukaryotic chromosomes that are composed of specific repeat DNA sequences and binding proteins [1]. Besides its function on telomeric DNA elongation, the telomerase contributes to telomere capping [16]. Another key regulator of telomere homeostasis is the Cdc13-Stn1-Ten complex, which is involved in both telomere length regulation and telomere end protection. The deletion of the PIF1 gene causes mitochondrial defect and telomere elongation [36, 37], and the telomere lengthening is TLC1 (the gene encodes the RNA subunit of telomerase)-dependent [32, 33]. Our genetic and biochemical data suggest that Def1p is involved in telomere length regulation
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